It is now well established that cellular interactions are at least in part mediated by receptor/ligand interactions. An important class of receptors is a family of three calcium-dependent mammalian lectins, known as L, P and E-selectins, that have the ability to mediate the early steps of recruitment of leukocytes from blood stream in a variety of normal and pathologic situations. All three selectins recognize carbohydrate-based ligands such as sialyl lewis.sup.x (S Le.sup.x), sialyl lewis.sup.a, sulfated lewis.sup.x and sulfated lewis.sup.a type of structures. Several natural ligands of the selectins have been described, many of which share the biochemical properties of sialomucins. Recently, efforts have been directed towards the production of artificial selectin ligands, including compounds that mimic the sialyl lewis.sup.x structure. However, the affinity of selectins to such synthetic analogs was poor when compared to that of the natural glycoprotein ligands (such as GlyCAM-1, CD34), the mucosal addressin-cell adhesion molecule-1 (MadCAM-1), and an as yet unidentified 200 kDa glycoprotein ligand (Rosen, S. D. et al. Curr. Opin. Cell Biology 6:663-673 (1994)). Rosen and co-workers demonstrated that the O-glycans of GlyCAM-1 are sialylated, fucosylated and sulfated, and that all three components are important for binding. It is surprising that PSGL-1 from HL-60 cells is not heavily fucosylated, and a majority of O-glycans are disialylated or neutral forms of Core 2 structures. A monosialylated trifucosylated glycan with a polylactosamine backbone at the C-6 of GalNAc in the Gal.beta.1.fwdarw.3GalNAc has also been reported as a minor constituent of PSGL-1.
In a recent study, it was observed that the compound NeuAc.alpha.2.fwdarw.3SE-6 Gal.beta.1.fwdarw.4(Fuc.alpha.1.fwdarw.3)GlcNAcOMe, which is pall of GlyCAM-1, was not a superior ligand for L-selectin. Both this compound and NeuAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.4(Fuc.alpha.1.fwdarw.3)SE-3GlcNAc. beta.1.fwdarw.3Gal, which was used by Scudder and co-workers in inhibition studies (Scudder, P. R., et al., Glycobiol. 44:929.fwdarw.933 (1994)), lacked a high affinity for L-selectin. As mentioned earlier, many of the selectin natural ligands are sialomucin type (or O-linked glycans). However, an extended trimannosyl oligosaccharide (N-linked type glycan) containing GalNAc Le.sup.x [GalNAc.beta.1.fwdarw.4(Fuc.alpha.1.fwdarw.3)GlcNAc.beta.1.fwdarw.Man.alph a.1.fwdarw.3/6Man.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw.4GlcNAc] was isolated from human cell produced Protein C, and shown to be a potent inhibitor for E-selectin.
Several theories have been postulated to explain the disparity between the high affinities of the selectins for their natural ligands and the relatively poor affinities for the sialylated/sulfated fucosylated lactosamines. Simple multivalency of both oligosaccharide and selectin on intact cell surfaces or by presentation on a polypeptide backbone could enhance avidity. This is apparent from comparison studies of the IC.sub.50 of monomeric, dimeric and tetravalent forms of SLe.sup.x for E-selectin, with the latter showing considerable improvement in inhibiting L-selectin. Nonetheless, P- and L-selectins do not bind to some cell types that express considerable amounts of SLe.sup.x, and cell recognition is usually destroyed by mucin-degrading enzyme O-sialoglycoprotease, even when the vast majority of cell surface SLe.sup.x remains intact after this treatment, indicating the insufficiency of simple ligand multivalency for explaining biologically relevant selectin binding. Multivalent aggregation of selectins could also be invoked. However, the high affinity binding of soluble monomeric E- and P-selectin to cell surfaces, indicates that this is not essential. Moreover, there is no published evidence thus far for naturally occurring multimerization of selecting. Similarly, the possibility of multiple binding sites within a single selectin lectin domain is unlikely, based on studies which indicate that the binding sites for carbohydrates are quite small. Alternatively, it could be hypothesized that the natural selectin ligands carry rare structural variants of the common sialylated fucosylated oligosaccharides which are responsible for the high affinity interaction. Because of the role of selectins in disease, particularly diseases involving undesired cell-cell adhesion that occurs through selectin-ligand binding on defined cell types, the identification and procurement of novel ligands that would allow the regulation of this type of selectin-ligand binding is greatly needed.